Sequence-specific DNA binding of transcription factors (TFs) is the foundation of regulated gene expression. Two recent observations have broadened the lexicon of sequence-specific DNA binding of mammalian TFs: the first is that methylated cytosines (5mC) are iteratively oxidized by the ten-eleven-translocation family of dioxygenases to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). 5fC and 5caC are removed by thymine DNA glycosylase, completing the DNA demethylation cycle. However, 5hmC is found accumulatesd in several tissues suggesting it may have a regulatory function. The second finding is that 5mC can occur outside of CG dinucleotides, initially being observed in stem cells and later in the brain. 5mC in CG dinucleotides is known to inhibit or enhance the DNA binding of B-ZIP proteins. The cAMP response element-binding protein (CREB1) regulates expression of cellular genes by binding to its consensus CRE sequence (TGAC'GTCA) and its variants, including the CRE/CEBP chimeric site (TGAC'GCAA). Double-stranded cytosine methylation of the CG dinucleotide at the center of the CRE motif inhibits CREB1 binding, leading to suppression of gene expression. In contrast, methylation of the central CG dinucleotide in the C/EBP motif TTGC'GCAA increases DNA binding of C/EBP family members. While the effect of 5mC and 5hmC in CG dinucleotides on sequence-specific DNA binding of transcription factors has been explored, their effect outside of CG dinucleotides on DNA binding has not been examined. The B-ZIP family of transcription factors have a bipartite structure with the leucine zipper region mediating dimerization and the N-terminal basic region mediating sequence specific DNA binding. The structural determinants of leucine zipper domain mediated B-ZIP dimerization specificity and stability have been investigated in detail. However, the relationship between the amino acid sequence of the B-ZIP basic region and the DNA sequences they bind, including those with modified cytosines, has not been explored. T7 DNA polymerase can efficiently incorporate 5mC and 5hmC into DNA when double-stranding single-stranded DNA. We exploited this property to double-strand single-stranded DNA 60-mers on an Agilent microarray using 5mC or 5hmC, creating double-stranded DNA sequences containing an asymmetric distribution of 5mC and 5hmC that mimics what occurs in several cell types in vivo. Using these protein binding microarrays, containing either cytosines on both DNA strands or a combination of cytosines on one strand and 5mC or 5hmC on the second strand, we started to examined the sequence specific binding of a number of B-ZIP and helix-loop-helix transcription factors in addition to CREB1: i.e., ATF1 C/EBP, JUND, TCF4, USF1 and ZTA.